Methods and Compositions for the Rapid Synthesis of Radiometal-Labeled Probes

ABSTRACT

The present application is directed to rapid and efficient synthesis of radiometal-labeled probes, pharmaceutical compositions comprising radiometal-labeled probes, and methods of using the radiometal-labeled probes. Such radiometal-labeled probes can be used in imaging studies, such as Positron Emitting Tomography (PET) or Single Photon Emission Computed Tomography (SPECT), and therapy studies.

FIELD OF THE INVENTION

The present invention is generally directed to method and compositions for the rapid synthesis of radiometal-labeled probes.

BACKGROUND OF THE INVENTION

Radiopharmaceuticals are radioactive drugs which can have either diagnostic or therapeutic applications. In diagnostic nuclear medicine, the radionuclide is used as a tracer to provide functional information about the tissue under investigation [1-2]. In contrast, therapeutic radionuclides should have particulate radiations with an in vivo range sufficient to destroy the target tissue but not so long as to cause collateral damage. The nuclear requirements for either diagnosis or therapy are seldom met by the radionuclides of the biologically ubiquitous elements such as C, H, N, O, etc., but oblige the use of biologically unimportant elements, almost all of which are metallic. For example, several non-conventional metallic radionuclides, such as ⁶⁴Cu, ⁸⁶Y, and ⁸⁹Zr, have been applied to positron emission tomography (PET) probes [3]. These metallic PET isotopes are usually characterized by longer half-lives, allowing the evaluation of radiopharmaceutical kinetics in same subject to be achieved by successful PET imaging over several hours or even days. Among these metallic radionuclides, ⁶⁴Cu (t_(1/2)=12.7 h; β⁺ 655 keV, 17.8%) has attracted considerable interest because of its favorable decay half-life, low β⁺ energy, and commercial availability [4].

Click chemistry offers chemists a platform for general, modular and high yielding synthetic transformations for constructing highly diverse molecules [5]. The Huisgen 1,3-dipolar cycloaddition reaction, which fuses an azide and an alkyne together, and provides access to a variety of five-membered heterocycles, has become of great use in labeling studies, the development of new therapeutics and nanoparticles, and in protein modification [6]. However, the Huisgen 1,3-dipolar cycloaddition reaction often requires the presence of catalytic amounts of non-radiolabeled Cu(I) ions, which interfere with radiometals and make click reaction impossible for the direct construction of radiometal-labeled probes.

To the best of our knowledge, the catalyst-free click reaction has not yet been employed in the radiometal-labeled probes.

SUMMARY OF THE INVENTION

One embodiment of the present invention is directed to a rapid, efficient, and catalyst-free method for generating radiometal-labeled probes and method of imaging integrin receptors (specifically, Integrin α_(v)β₃) in vivo with the radiometal-labeled cyclopeptide derivatives.

Another embodiment of the present invention utilizes a Huisgen 1,3-dipolar cycloaddition reaction including an aza-dibenzocyclooctyne ligation to assemble radiometal-labeled probes with various hydrophilic linkages, such as polyethelene glycol units and other sugar mimetics. This synthetic method is characterized by its rapidity (<15 min) and excellent yield (>98%) without adding of any catalysts. It is not possible to use a catalyst-free methodology for preparing radiometal-labeled probes using conventional Huisgen 1,3-dipolar cycloaddition reaction. The newly developed cyclopeptide derivatives obtained using the methods described herein maintain good binding affinity to integrin α_(v)β₃ receptor, and have excellent tumor uptake and favorable pharmacokinetic behavior.

Another embodiment of the present invention is a fast and efficient for radiometal-labeling using catalyst-free click chemistry, and its application to the synthesis of a ⁶⁴Cu-labeled derivative of cyclic RGD peptide [c(RGDfK)](FIG. 1), an integrin α_(v)β₃ ligand [8]. The so-called “click” probe maintains good binding affinity to integrin α_(v)β₃ receptor, and exhibits excellent tumor targeting and retention properties in an integrin α_(v)β₃-positive mouse tumor model.

Another embodiment of the present invention is a new catalyst-free click chemistry approach based on aza-dibenzocyclooctyne ligation has been developed for radiometal-labeling of biomolecules. The reaction proceeds with fast rates, making it an attractive radiometal-labeling method at low concentration. Our technique is novel and general, and can be applied to other radiometal-labeled probes for imaging and therapy applications.

One embodiment of the present invention is directed to a rapid, efficient, and catalyst-free click chemistry method for generating radiometal-labeled probes for imaging and therapy according to Formula III comprising: reacting an azido composition, N₃-L (Formula I, below) with a strained alkyne (Formula II, below) as shown in the following Reaction (Reaction 1):

wherein L may be conjugated to any of the nitrogen atoms of the triazole moiety, wherein L includes at least one biological component, including without limitation a small molecule, peptide, protein and/or nanoparticle, or alternatively, L is a chelator capable of coordinating a radiometal nuclide,

the moiety M is an optional linker moiety, but when included may comprise one or more hydrophilic groups, including, for example, one or more polyethelene glycol units, one or more sugars,

the moiety Q is a chelator capable of coordinating a radiometal nuclide, or alternatively, if L is a chelator, Q includes at least one biological component, including without limitation a small molecule, peptide, protein and/or nanoparticle, and

Another embodiment of the present invention is directed to a cyclopeptide derivative of the following Formula IV.

wherein at least on of moieties X, Y and Z includes a click chemistry linkage, such as a triazole moiety,

wherein R is selected from the group consisting of a side chain of a natural amino acid and a side chain of an unnatural amino acid, wherein the natural amino acid and the unnatural amino acid is either in the D or L form;

wherein X is a heterocycle or a linker comprising a hydrophilic moiety selected from the group consisting of hydroxyl, carbonyl, sulfonamide, sulfonate, phosphate, polar amino acid moiety, PEG moiety, sugar mimetic, and sugar moiety;

wherein Y is a heterocycle, or a linker comprising a hydrophilic moiety selected from the group consisting of hydroxyl, carbonyl, sulfonamide, sulfonate, phosphate, polar amino acid moiety, PEG moiety, sugar mimetic, and sugar moiety;

wherein Z is a heterocycle, or a linker comprising a hydrophilic moiety selected from the group consisting of hydroxyl, carbonyl, sulfonamide, sulfonate, phosphate, polar amino acid moiety, PEG moiety, sugar mimetic, and sugar moiety;

wherein w is 1, 2, 3, 4, or 5; and

wherein any one of X, Y, and Z comprises a radiometal nuclide, including, but not limited to the following: ⁶¹Cu, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ⁶⁸Ga, ⁸⁶Y, and ⁸⁹Zr. In a first preferred embodiment, Y is a heterocycle; and X is a linker either comprising a sugar mimetic selected from the group consisting of a 4 to 6-membered carbocycle substituted with at least one hydroxyl group and a 5- to 6-membered heterocycle substituted with at least one hydroxyl group or comprising a sugar moiety selected from the group consisting of glucose and galactose.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates the chemical structures of several RGD peptides.

FIG. 2 shows the (a) Synthesis of [⁶⁴Cu]1. Reagents and conditions: (i) ⁶⁴CuCl₂, 40° C., 0.4 M NH₄OAc; (ii) RGD peptide 2, 45° C., H₂O; (iii) RGD peptide 2, room temperature, H₂O; (iv) ⁶⁴CuCl₂, 0.4 M NH₄OAc. (b) Radio-HPLC profiles of [⁶⁴Cu]1 and [⁶⁴Cu]3.

FIG. 3 shows the stability of [⁶⁴Cu]1 in PBS (pH=7.4) at room temperature for 1, 6, and 24 h.

FIG. 4 shows the in vitro inhibition of ¹²⁵I-echistatin bound to integrin α_(v)β₃ on U87MG glioma cells by the non-modified peptide c(RGDyK) and peptide 1. The IC₅₀ values of c(RGDyK) and peptide 1 were calculated to be 105±5 nM and 170±3 nM, respectively.

FIG. 5 shows (a) decay-corrected whole-body microPET images of U87MG tumor bearing mice (n=3) at 2, 4, 20 h after intravenous injection of [⁶⁴Cu]1. The image obtained with co-injection of c(RGDyK) (10 mg/kg body weight) is shown for 2 h blockade (right). Tumors are indicated by arrows. (b) MicroPET quantification of tumors and major organs at 2, 4, and 20 h after intravenous injection of [⁶⁴Cu]1. The blockade with co-injection of c(RGDyK) (10 mg/kg body weight) is only shown at 2 h post injection.

FIG. 6 shows the biodistribution of [⁶⁴Cu]1 in U87MG tumor bearing athymic nude mice at 20 h with and without co-injection of 10 mg/kg of c(RGDyK) as a blocking agent (n=3/group).

DETAILED DESCRIPTION OF THE INVENTION Definitions

The term “alkyl” means a straight or branched chain acyclic saturated hydrocarbon. The term “C₁-C₆ alkyl” herein used means C₁-C₆ straight or branched chain alkyl, for example, methyl, ethyl, n-propyl, i-propyl, n-butyl, butyl, sec-butyl, tert-butyl, n-pentyl, i-pentyl, neo-pentyl, tert-pentyl, and the like.

The term “alkenyl” refers to a branched or unbranched hydrocarbon having at least one carbon-carbon double bond. The term “C₂₋₆-alkenyl” refers to a hydrocarbon having two to seven carbon atoms and comprising at least one carbon-carbon double bond.

The term “alkynyl” refers to a branched or unbranched hydrocarbon having at least one carbon-carbon triple bond. The term “C₂₋₆-alkynyl” refers to a hydrocarbon having two to seven carbon atoms and comprising at least one carbon-carbon triple bond.

The term “carbocycle” refers to a non-aromatic stable 3- to 8-membered carbon ring which may be saturated, mono-unsaturated or poly-unsaturated. The carbocycle may be attached at any endocyclic carbon atom which results in a stable structure. Preferred carbocycles have 5-7 carbons.

The term “aryl” herein used means monocyclic or condensed ring aromatic hydrocarbons. Examples of the aryl are phenyl, naphthyl, and the like.

The term “heterocycle” as used herein is intended to mean monocyclic, spirocyclic, bicyclic or tricyclic ring of up to 10 atoms in each ring, wherein each ring is aromatic or non-aromatic and contains carbon and from 1 to 4 heteroatoms selected from the group consisting of O, N, P and S. A nonaromatic heterocycle may be fused with an aromatic aryl group such as phenyl or aromatic heterocycle.

The term “halogen” herein used means fluoro, chloro, bromo, and iodo.

The term “hydroxy” as used herein, means an —OH group.

The term “nitro” as used herein, means a —NO₂ group.

The term “carbonyl” as used herein, means a—C(O)— group.

The term “sulfonamide” refers to the group —SO₂NH₂.

The term “sulfonic acid” and “sulfonate” refers to the group SO₃H, or a and an acceptable salt or ester thereof, respectively.

The term “phosphate” refers to an ester of phosphoric acid, and includes salts of a phosphate moiety.

The term “amine” includes primary, secondary, and tertiary amines having, e.g., the formula N(group)3 wherein each group can independently be H or non-H, such as alkyl, aryl, and the like. Amines include but are not limited to RNH₂, for example, alkylamines, arylamines, alkylarylamines; R2NH wherein each R is independently selected, such as dialkylamines, diarylamines, aralkylamines, heterocyclylamines and the like; and R3N wherein each R is independently selected, such as trialkylamines, dialkylarylamines, alkyldiarylamines, triarylamines, and the like. The term “amine” also includes ammonium ions as used herein.

The term “alkoxy” denotes straight chain or branched alkoxy, preferably C₁₋₆ alkoxy. Examples of alkoxy include methoxy, ethoxy, n-propoxy, isopropoxy and the different butoxy isomers.

The term “optionally substituted” means the addition of a substituent group to the composition that is referred to. Suitable substituents include hydroxy, alkoxy (e.g., methoxy and ethoxy), mercapto, alkylthio (e.g., methylthio), cycloalkyl (e.g., cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl), halogen (e.g., fluoro, chloro, bromo, and iodo), carboxy, alkoxycarbonyl (e.g., methoxycarbonyl and ethoxycarbonyl), nitro, cyano, haloalkyl (e.g., trifluoromethyl), substituted or unsubstituted amino (e.g., methylamino, dimethylamino, and carbamoylamino), guanidino, phenyl, benzyloxy, and the like. These substituents are able to bind them at one or more of any possible positions

As used herein, the term “hydrophilic moiety,” is any anionic, cationic, zwitterionic, or nonionic group that is polar. Nonlimiting examples include anionics such as sulfate, sulfonate, carboxylic acid/carboxylate, phosphate, phosphonates, and the like; cationics such as: amino, ammonium, including mono-, di-, and trialkylammonium species, pyridinium, imidazolinium, amidinium, poly(ethyleneiminium), and the like; zwitterionics such as ammonioalkylsulfonate, ammonioalkylcarboxylate, amphoacetate, and the like; and nonionics such as hydroxyl, sulfonyl, ethyleneoxy, amido, ureido, amine oxide, and the like.

The term “sugar moiety” means monosaccharides (e.g., glucose, arabinose, fucose, galactose, mannose, xylose, fructose, lyxose, allose, arinose, ribose, talose, gulose, idose, altrose, sorbitol, mannitol or glucosamine), disaccharides and oligosaccharides (e.g., maltose, isomaltose, turanose, gentiobiose, melibiose, planteobiose, primererose, vicianose, nigerose, laminaribiose, rutinose, cellobiose, xylobiose, maltotriose, gentianose, melezitose, planteose, ketose, trehalose, sucrose, lactose, raffinose or xylobiose), polysaccharides (e.g., amylose, ficol, dextrin, starch, dextran, polydextrose, pullulan, cyclodextrin, glucomannoglycan, glucomannan, guar gum, gum arabic or glycosaminoglycan), complex carbohydrates (e.g., glycopeptide, glycoprotein, glycolipid or proteoglycan), and the like.

The term “PEG” is used broadly to encompass any polyethylene glycol molecule, without regard to size or to modification at an end of the PEG, and can be represented as linked to the FGF-21 polypeptide by the formula: XO—(CH₂CH₂O)_(n)—CH₂CH₂—Y where n is 2 to 10,000 and X is H or a terminal modification, including but not limited to, a C₁₋₆ alkyl, a protecting group, or a terminal functional group.

As used herein, the term “PEG moiety” is intended to include, but is not limited to, linear and branched PEG, methoxy PEG, hydrolytically or enzymatically degradable PEG, pendant PEG, dendrimer PEG, copolymers of PEG and one or more polyols, and copolymers of PEG and PLGA (poly(lactic/glycolic acid)) and the like. According to the present invention, the term polyethylene glycol or PEG is meant to comprise native PEG as well as derivatives thereof.

The term “natural amino acid” refers to one of the twenty commonly occurring amino acids. Natural amino acids can be in their D or L form. For example, a natural amino acid can be selected from the group consisting of L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-glutamic acid, L-glutamine, L-glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, and mixtures thereof. In some cases, an amino acid is selected from L-glycine and L-aspartic acid. The term “unnatural amino acid” is any amino acid that is not a natural amino acid.

As used herein, the term “polar amino acid moiety” refers to the side chain of a polar natural or unnatural amino acid. Polar natural amino acids include, but are not limited to, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, histidine and lysine.

The term “peptide” is a sequence of 2 to 25 amino acids (e.g. as defined hereinabove) or peptidic residues having one or more open valences. The sequence may be linear or cyclic. For example, a cyclic peptide can be prepared or may result from the formation of disulfide bridges between two cysteine residues in a sequence. A peptide can be linked through the carboxy terminus, the amino terminus, or through any other convenient point of attachment, such as, for example, through the sulfur of a cysteine. Peptide derivatives can be prepared as disclosed in U.S. Pat. Nos. 4,612,302; 4,853,371; and 4,684,620. Peptide sequences specifically recited herein are written with the amino terminus on the left and the carboxy terminus on the right.

The term “polypeptide” refers to a biopolymer compound made up of a single chain of amino acid residues linked by peptide bonds. The term “protein” as used herein may be synonymous with the term “polypeptide” or may refer, in addition, to a complex of two or more polypeptides.

As used herein, reference to “Arg-Gly-Asp peptide” or “RGD peptide” is intended to refer to a peptide having one or more Arg-Gly-Asp containing sequence which may function as a binding site for a receptor of the “Arg-Gly-Asp family of receptors”, e.g., an integrin. The sequence RGD is present in several matrix proteins and is the target for cell binding to matrix by integrins. Platelets contain a large amount of RGD-cell surface receptors of the protein GP II_(b)/III_(a), which is primarily responsible, through interaction with other platelets and with the endothelial surface of injured blood vessels, for the development of coronary artery thrombosis. The term RGD peptide also includes amino acids that are functional equivalents (e.g., RLD or KGD) thereof provided they interact with the same RGD receptor. Peptides containing RGD sequences can be synthesized from amino acids by means well known in the art, using, for example, an automated peptide synthesizer, such as those manufactured by Applied Biosystems, Inc., Foster City, Calif.

The term “chelate” may be used as a noun or a verb. As a noun, “chelate” refers to one or more atoms that are either capable of chelating one or more metal ions, or are chelating to one or more metal ions. In some embodiments, only one metal ion coordinates to a chelate. A chelator refers to compound comprising a chelate that is a) capable of chelating to one or more metal ions or b) are coordinated to (or chelated to) to one or more metal ions. Typically, just one metal ion is chelated to a chelator.

The term “linker” means any chemical group positioned between different segments of the compounds of the present invention. These chemical groups may be of any stable chemical structure, and may provide spacing between segments, impart conformational constraints on the segments, provide drug targeting or drug delivery functionality, increase absorption and/or lifespan in vivo, or provide any additional ancillary role that benefits the utility of the molecule. Examples include, but are not limited to, methylene —(CH₂)_(n)— units, polyethylene glycol or other bioploymeric molecules, sugar or moieties, natural products, peptide-nucleic acid (PNA) molecules, both natural and unnatural amino acids and nucleic acids.

A copper-free click reaction was reported by Ning et al. (2010, Angew Chem Int; Ed 49:3065-68), involving strain-promoted alkyne-nitron cycloaddition. To address the slow rate of the original cyclooctyne reaction, electron-withdrawing groups are attached adjacent to the triple bond (Id.) Examples of such substituted cyclooctynes include difluorinated cyclooctynes, 4-dibenzocyclooctynol and azacyclooctyne (Id.)

One embodiment of the present invention is directed to a rapid, efficient, and catalyst-free click chemistry method for generating radiometal-labeled probes for imaging and therapy according to Formula III comprising: reacting an azido composition, N₃-L (Formula I, below) with a strained alkyne (Formula II, below) as shown in the following Reaction (Reaction Scheme 1):

wherein L may be conjugated to any of the nitrogen atoms of the triazole moiety, wherein L includes at least one biological component, including without limitation a small molecule, peptide, protein and/or nanoparticle, or alternatively, L is a chelator capable of coordinating a radiometal nuclide,

the moiety M is an optional linker moiety, but when included may comprise one or more hydrophilic groups, including, for example, one or more polyethelene glycol units, one or more sugars,

the moiety Q is a chelator capable of coordinating a radiometal nuclide, or alternatively, if L is a chelator, Q includes at least one biological component, including without limitation a small molecule, peptide, protein and/or nanoparticle, and

In Formula II, the benzene rings may be mono-, di-, tri-, or tetra-substituted with R₁ and R₂ groups selected from the group consisting of H, halide, hydroxy, alkyl, aryl, nitro or amine groups. In a preferred embodiment, chelator is chelated to a radiometal nuclide which may including, but is not limited to the following: ⁶¹Cu, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ⁶⁸Ga, ⁸⁶Y, and ⁸⁹Zr.

Another embodiment of the present invention is directed to a cyclopeptide derivative of the following Formula IV.

wherein at least on of moieties X, Y and Z includes a click chemistry linkage, such as a triazole moiety,

wherein R is selected from the group consisting of a side chain of a natural amino acid and a side chain of an unnatural amino acid, wherein the natural amino acid and the unnatural amino acid is either in the D or L form;

wherein X is a heterocycle or a linker comprising a hydrophilic moiety selected from the group consisting of hydroxyl, carbonyl, sulfonamide, sulfonate, phosphate, polar amino acid moiety, PEG moiety, sugar mimetic, and sugar moiety;

wherein Y is a heterocycle, or a linker comprising a hydrophilic moiety selected from the group consisting of hydroxyl, carbonyl, sulfonamide, sulfonate, phosphate, polar amino acid moiety, PEG moiety, sugar mimetic, and sugar moiety;

wherein Z is a heterocycle, or a linker comprising a hydrophilic moiety selected from the group consisting of hydroxyl, carbonyl, sulfonamide, sulfonate, phosphate, polar amino acid moiety, PEG moiety, sugar mimetic, and sugar moiety;

wherein w is 1, 2, 3, 4, or 5; and

wherein any one of X, Y, and Z comprises a radiometal nuclide, including, but not limited to the following: ⁶¹Cu, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ⁶⁸Ga, ⁸⁶Y, and ⁸⁹Zr. In a first preferred embodiment, Y is a heterocycle; and X is a linker either comprising a sugar mimetic selected from the group consisting of a 4 to 6-membered carbocycle substituted with at least one hydroxyl group and a 5- to 6-membered heterocycle substituted with at least one hydroxyl group or comprising a sugar moiety selected from the group consisting of glucose and galactose.

In a second preferred embodiment, Y is a heterocycle;

-   -   X is selected from the group consisting of:

Z is selected from the group consisting of:

wherein T is selected from the group consisting of:

wherein W is selected from the group consisting of:

wherein E is selected from the group consisting of:

each R is independently selected from the group consisting of a side chain of a natural amino acid and a side chain of an unnatural amino acid, wherein the natural amino acid and the unnatural amino acid is either in the D or L form;

each R₃ is independently selected from the group consisting of —H, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ alkyloxy, aryl, aryl-(C₁-C₆ alkylene)-, 3- to 7-membered carbocycle, 3- to 7-membered heterocycle, hydroxy-C₁-C₆-alkyl, C₁-C₆-alkoxy-C₁-C₆-alkyl, and a PEG moiety, wherein the alkyl, alkenyl, alkynyl, alkoxy, aryl, carbocycle, and heterocycle groups are each optionally substituted;

each R₄ is selected from the group consisting of —H, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ alkyloxy, aryl, aryl-(C₁-C₆ alkylene)-, 3- to 7-membered carbocycle, 3- to 7-membered heterocycle, hydroxy-C₁-C₆-alkyl, and C₁-C₆-alkoxy-C₁-C₆-alkyl, wherein the alkyl, alkenyl, alkynyl, alkyloxy, aryl, carbocycle, and heterocycle, groups are each optionally substituted;

each R₅ is independently selected from the group consisting of —H, —OH, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ alkyloxy, aryl-(C₁-C₆ alkylene)-, hydroxy-C₁-C₆-alkyl, and C₁-C₆-alkoxy-C₁-C₆-alkyl, wherein the alkyl, alkenyl, alkynyl, alkyloxy, and aryl-alkylene groups are each optionally substituted;

each v is 0, 1, 2, 3, or 4;

m is 0, 1, 2, 3 or 4;

p is an integer between 1 and 110;

q is 1, 2, 3 or 4;

r is 1, 2 or 3;

r′ is 0 or 1;

s is 1, 2, 3 or 4; and

the radionuclide is selected from a radiometal nuclide, including, but not limited to the following: ⁶¹Cu, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ⁶⁸Ga, ⁸⁶Y, and ⁸⁹Zr.

wherein the configuration of the chiral centers may be R or S or mixtures thereof.

In another preferred embodiment, R is a side chain of a natural amino acid;

X is

Wherein the variables T, E, W;

Y is 3a,8,9,13b-tetrahydro-3H-dibenzo[b,f][1,2,3]triazolo[4,5-d]azocine; and

Z is selected from the group consisting of:

R₃ is selected from the group consisting of —H, C₁-C₆ alkyl, C₂-C₆ alkenyl, and C₂-C₆ alkynyl, wherein the alkyl, alkenyl, and alkynyl groups are each optionally substituted; and any one of X, Y, and Z comprises a radiometal nuclide including, but not limited to the following: ⁶¹Cu, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ⁶⁸Ga, ⁸⁶Y, and ⁸⁹Zr.

Preferably, a portion of the peptide contains a primary amine group, such as a lysine residue, which is utilized for conjugation to moiety XYZ. The configuration of C-α of the amine-containing residue may be R or S. Preferably, the moiety XYZ contains zero, one, or more hydrophilic groups (such as hydroxyl, sulfonate, phosphate, etc.).

Preferably, the nuclide label A is covalently conjugated to one or more of X, Y, and Z.

Another embodiment of the present invention is directed to a method for preparing a radiopharmaceutical comprising reacting an azido compound formula N₃-L with a strained alkyne to form of a 1,2,3-triazole under reaction conditions that do not include a catalyst. Preferably, the strained alkyne has a formula according to Formula II, and the benzene rings may be mono-, di-, tri-, or tetra- substituted with R₁ and R₂ groups selected from the group consisting of H, halide, hydroxy, alkyl, aryl, nitro or amine groups. Preferably, L is cyclopeptide and the strained alkyne is capable of being coupled to a chelator. Preferably, the strained alkyne is coupled to a chelator prior to reaction with the azido compound to form an alkyne-containing chelator. Preferably, the alkyne-containing chelator is labeled by a radiometal prior to reaction with the azido compound. The radiometal may be selected from the group consisting of ⁶¹Cu, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ⁶⁸Ga, ⁸⁶Y, and ⁸⁹Zr.

Another embodiment of the present invention is a method of modifying an RGD-sequence containing cyclic peptide with the moiety XYZ, which installs a nuclide label on the cyclic peptide, comprising: click chemistry.

A catalyst-free click chemistry approach in accordance with the present invention, describes the rapid, selective and specific formation of 1,2,3-triazoles starting from an azide and a strained alkyne moiety. An azido group can be attached to the cyclopeptide while a strained alkyne moiety can be coupled with a chelator. Afterwards, the alkyne-containing chelator can be labeled by a radiometal, and followed by a rapid assembling with azido-containing piece to form the final radiometal-labeled probes without adding of any catalysts. The hydrophilic linkages can also be readily incorporated into the final product. Because of this high-yielding and modular approach, the pharmacokinetic properties of final radiometal-labeled probes are easily modified.

One embodiment of the present invention is a catalyst-free click chemistry approach to rapidly yet efficiently construct a ⁶⁴Cu-labeled cyclopeptide for PET imaging of integrin α_(v)β₃ receptor in vivo. Various aspects and embodiments are discussed in relation to the ⁶⁴Cu-labeled cyclopeptide described herein, and other aspects and embodiments will be apparent to those skilled in the art. The various aspects and embodiments disclosed herein are for purposes of illustration and are not intended to be limiting. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the method and compositions described.

In a preferred embodiment, a suitable azide moiety containing RGD peptide is selected. One of ordinary skill in the art can modify the RGD peptide according a particular application. For instance, in one embodiment, it was found that glycosylation on the lysine side chain of cyclic RGD peptides decreased lipophilicity and hepatic uptake [9]. This finding prompted us to consider methods for preparing azido galacto-RGD peptide. To this end, compound 2 as shown in FIG. 1. The fully protected c[R(Pbf)GD(O^(t)Bu)fK] peptide was conjugated with Fmoc-protected galactose, followed by deprotection of the Fmoc group, azido acetic acid coupling, and guanidine and acid deprotections. The synthesis was achieved in four steps with a total yield of 44%. Compound 2 was obtained in a great chemical purity (>95%) without HPLC purification.

In a preferred embodiment, a suitable ⁶⁴Cu-chelator complex system, which can be readily conjugated with a strained alkyne should be selected. Suitable bifunctional chelators useable with the invention include oxygen and nitrogen containing cyclic chelators known to those of ordinary skill. Various bifunctional chelators (BFCs) are useable in connection with the present invention, including cyclam ((1,4,8,11-tetraazacyclotetradecane) and cyclen (1,4,7,10-tetraazacyclododecane) backbone-based chelators and cross-bridged tetraamine ligands, all of which have been developed for ⁶⁴Cu labeling [4, 10]. Suitable BFCs include cage-like hexaazamacrobicyclic sarcophagine (Sar), has gained great attention as potential ⁶⁴Cu chelators. We and others demonstrated that either one of the inert primary amines of 3,6,10,13,16,19-hexaazabicyclo[6.6.6]eicosane-1,8-diamine (DiAmSar) or both primary amines could be modified and coupled with biologically relevant ligands[11]. The resulting ⁶⁴Cu complexes present improved in vivo stability and radiolabeling efficiency. On the other hand, an aza-dibenzocyclooctyne system has been proved to be simultaneously reactive and stable.[12]

In one embodiment, a DiAmSar-containing dibenzocyclooctyne analog as a strained alkylne as well as a ⁶⁴Cu labeling precursor was built. A short polyethylene glycol) (PEG) linker was introduced between DiAmSar and dibenzocyclooctyne to fine-tune the in vivo pharmacokinetics of the resulting probes. Our synthesis started from commercially available dibenzocyclooctyne, which was coupled with a short PEG linker. After the activation of carboxylic acid group, the PEG-dibenzocyclooctyne was conjugated to commercially available DiAmSar in basic sodium borate buffer to afford 3 in 45% yield. Radiolabeling of 3 with ⁶⁴Cu was efficiently accomplished at 40° C. in 0.4 M NH₄OAc buffer within 30 min (FIG. 2 a). The labeled product [⁶⁴Cu]3 was purified by HPLC. The radioactive peak containing [⁶⁴Cu]3 appeared at 17.67 min as shown in FIG. 2 b. The specific activity of [⁶⁴Cu]3 was estimated to be 37 MBq nmol⁻¹.

The conjugations between 2 and [⁶⁴Cu]3 were carried out in deionized water and analyzed by analytical HPLC. When [⁶⁴Cu]3 (3.7 Mbq, 1 μM) was mixed with a large excess of 2 (>100 folds) at room temperature, [⁶⁴Cu]3 was rapidly consumed within 10-15 min, and [⁶⁴Cu]1 (R_(t)=15.05 min, FIG. 2 b) formed in >92% radiochemical yield (Table 1, entries 1 and 2). With a small excess of 2 (5.7 folds), the radiochemical yield was decreased to 42% after combining 2 and [⁶⁴Cu]3 for 10 min at room temperature (entry 3). However, the elevated temperature (45° C.) significantly enhanced the [⁶⁴Cu]1 yield (>98%, entry 4) after 10-min mixing of 2 and [⁶⁴Cu]3. With prolonged reaction time (15 min), an excellent radiochemical yield (>98%) of [⁶⁴Cu]1 was still achieved by using 1.14:1 ratio of 2 and [⁶⁴Cu]3 (entry 5). Further reducing the concentration of 2 and [⁶⁴Cu]3 resulted in a low [⁶⁴Cu]1 yield (16%, entry 6). We also investigated the efficiency of the conjugation between 2 and [⁶⁴Cu]3 in PBS buffer. As shown in entry 7, [⁶⁴Cu]1 was formed in a quantitative yield at 45° C. within 15 min.

TABLE 1 Optimization of synthesis of [⁶⁴Cu]1 through the strain-promoted catalyst-free conjugation between 2 and [⁶⁴Cu]3. Temper- Reaction Radiochemical Entry 2 [⁶⁴Cu]3^(a) Solvent ature time/min yield (%) 1  228 μM 3.7 MBq (1 μM) H₂O 25° C. 15 >98  2  114 μM 3.7 MBq (1 μM) H₂O 25° C. 10 92 3  5.7 μM 3.7 MBq (1 μM) H₂O 25° C. 10 42 4  5.7 μM 3.7 MBq (1 μM) H₂O 45° C. 10 >98  5 1.14 μM 3.7 MBq (1 μM) H₂O 45° C. 15 >98  6 0.29 μM  1.85 MBq (0.5 μM) H₂O 45° C. 10 16 7 1.14 μM 3.7 MBq (1 μM) PBS buffer 45° C. 15 >98  ^(a)The concentration was estimated based on the specific activity of [⁶⁴Cu]3 (37 MBq nmol⁻¹), taking into account a correction of radioactive decay.

The in vitro stability of [⁶⁴Cu]1 was evaluated in PBS buffer by radio-HPLC. Chromatographic results demonstrated no release of ⁶⁴Cu from the conjugate over a period of 24 h (FIG. 3). This high stability is attributed to Sar cage in the conjugate. In addition, it is known that the U87MG cell line over-expresses integrin α_(v)β₃ receptor [13]. Therefore, we used the U87MG cell line to measure the integrin α_(v)β₃ binding affinity of 1 by a competitive cell-binding assay [13], where ¹²⁵I-echistatin was employed as integrin α_(v)β₃-specific radioligand for competitive displacement. The IC₅₀ values of c(RGDyK) and 1, which represent their concentrations required to displace 50% of the ¹²⁵I-echistatin bound to the U87MG cells, were determined to be 105±5 nM and 170±3 nM, respectively (FIG. 4). The slightly decreased integrin α^(v)β₃ binding of 1 as compared to c(RGDyK) indicates a minimum impact of a long tail (containing galactose, triazole, and Sar moieties) on the binding of c(RGDfK) to integrin α_(v)β₃.

The in vivo tumor-targeting efficacy of [⁶⁴Cu]1 was evaluated in nude mice bearing U87MG human glioma xenograft tumors (n=3) by static microPET scans at 2, 4, and 20 h after tail-vain injection of [⁶⁴Cu]1. Representative decay-corrected coronal images at different time points were shown in FIG. 5 a. U87MG tumors were clearly visualized at all time points examined. Region-of-interest (ROI) analysis on microPET images showed the tumor uptake values were 4.96±0.65, 4.11±0.48, and 2.41±0.31% ID/g at 2, 4, and 20 h post injection, respectively (FIG. 5 b). At 2 h post injection, the tumor/liver, tumor/kidneys, and tumor/muscle ratios reached 2.82±0.76, 2.22±1.49, and 11.79±2.65, respectively. Consequently, the high tumor-to-nontumor ratios provided excellent contrast for PET imaging. Blocking experiment was conducted to confirm the integrin α_(v)β₃ specificity of [⁶⁴Cu]1. In the presence of a blocking dose (10 mg/kg) of c(RGDyK), the U87MG tumor uptake was reduced to the background level (0.71±0.28% ID/g) at 2 h post injection (FIGS. 5 a and 5 b). The uptake values of normal organs (e.g., liver, kidneys, and muscle) were also lower than those without coinjection of c(RGDyK) (FIG. 5 b). The ex vivo biodistribution of [⁶⁴Cu]1 was examined in U87MG tumor-bearing mice at 20 h pi with and without coinjection of c(RGDyK). The results were consistent with the quantitative analysis of PET imaging (FIG. 6).

The following Examples are provided in order to demonstrate and further illustrate certain embodiments and aspects of the present invention and are not to be construed as limiting the scope thereof.

Synthesis of Peptide 2

The synthesis of Peptide 2 (shown below) is summarized in Scheme 1.

Preparation of Peptide S1

To a solution of (3R,4R,5S,6S)-6-((((9H-fluoren-9-yl)methoxy)carbonylamino)methyl)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (301 mg, 0.702 mmol, 1.2 equiv) in DMF (5 mL) was added HOBt (95 mg, 0.702 mmol, 1.2 equiv) and EDC (135 mg, 0.702 mmol, 1.2 equiv). The mixture was stirred at room temperature for 2 hrs. The protected c(RGDfK) TFA salt (600 mg, 0.585 mmol, 1.0 equiv) and DIPEA (0.204 mL, 1.169 mmol, 2 equiv) in DMF (5 mL) was added to the reaction dropwise. The mixture was stirred overnight. To the mixture was then added EtOAc (160 mL), ultrasonicated for 30 min, and cooled to 0° C. A white solid precipitated from the solution. The solid was filtered, washed with water (10 mL) and ether (20 mL×2), and dried. To a round bottom flask containing the solid (650 mg, 0.491 mmol, 1 equiv) was added piperidine (2.15 g, 25.2 mmol). After overnight stirring, the reaction was concentrated under vacuum to remove piperidine. Acetonitrile (20 mL×3) was added to facilitate co-evaporation. The residue was dried under vacuum for 2 hrs. The white solid residue was then washed with ether (20 mL×3) under ultrasound. The residue solid was filtered and dried under vacuum overnight to afford peptide S1 (560 mg) in a total of 90% yield. ¹H NMR (d₆-DMSO, 400 MHz), δ: 8.42 (t, 1H), 8.08-8.06 (m, 2H), 8.02-7.98 (d, 3H), 7.60 (t, 1H), 7.43 (t, 1H), 7.51 (d, 1H), 7.26-7.11 (m, 6H), 4.72 (d, 1H), 4.59 (q, 1H), 4.51 (d, 1H), 4.42 (q, 1H), 4.10 (m, 1H), 4.00 (dd, 1H), 3.95-3.85 (m, 2H), 3.72 (s, 1H), 3.76-3.70 (m, 2H), 3.25-3.20 (m, 2H), 3.05-2.87 (m, 9H), 2.83-2.75 (m, 1H), 2.65-2.55 (m, 3H), 2.47-2.33 (m, 8H), 1.97 (s, 3H), 1.65-1.48 (m, 2H), 1.40-1.20 (m, 20H), 0.97-0.95 (m, 2H). ESI-MS: calculated for C₅₁H₇₆N₁₀O₁₅S: 1100.52; found: 1101.4 [M÷H]⁺.

Preparation of Peptide S2

To a solution of azido acetic acid (5.65% solution in DCM/THF, 1.56 g, 0.872 mmol, 2.5 equiv) in DMF (2 mL), was added HOBt (118 mg, 0.872 mmol, 2.5 equiv) and EDC (167 mg, 0.872 mmol, 2.5 equiv). The mixture was stirred at room temperature for 20 min. Peptide S1 (400 mg, 0.349 mmol, 1 equiv) in DMF (7.5 vol) was added along with DIPEA (0.152 mL, 0.872 mmol, 2.5 equiv) to the above mixture. The reaction was stirred at room temperature for additional 20 min. The reaction was quenched by addition of water (few drops). DMF was removed under vacuum. Acetonitrile (15 mL×3) was added to facilitate co-evaporation of DMF. To the residue was added water (90 mL) and treated with ultrasound for 30 min. The white solid precipitate was collected by filtration. The cake was washed with mother liquid (20 mL×1), ether (15 mL×2), and dried under vacuum with P₂O₅ overnight to afford peptide S2 (392 mg, 92% yield). ¹H NMR (d₆-DMSO, 400 MHz), δ: 8.42 (t, 1H), 8.11-8.05 (m, 2H), 8.00 (d, 2H), 7.50 (d, 1H), 7.43 (t, 1H), 7.26-7.11 (m, 5H), 4.92 (d, 1H), 4.78 (d, 1H), 4.59-4.53 (m, 2H), 4.42 (q, 1H), 4.10 (m, 1H), 4.00 (dd, 1H), 3.95-3.85 (m, 2H), 3.82 (d, 2H), 3.76-3.70 (m, 2H), 3.25-3.20 (m, 2H), 3.05-2.87 (m, 9H), 2.83-2.75 (m, 1H), 2.65-2.55 (m, 1H), 2.47-2.33 (m, 8H), 1.97 (s, 3H), 1.65-1.48 (m, 2H), 1.40-1.20 (m, 20H), 0.97-0.95 (m, 2H). ESI-MS: calculated for C₅₃H₇₇N₁₃O₁₆: 1183.53. found: 1184.4 [M+H]⁺.

Preparation of Peptide 2

To peptide S2 (40 mg, 0.034 mmol, 1 equiv) was added 2 mL of the following solvents TFA:TIS:H₂O=95:2.5:2.5. The reaction was stirred at room temperature for 30 min. To the mixture was added cold ether (25 mL). The precipitate was formed and concentrated after centrifugation. The precipitate was filtered and washed with cold ether (2 mL×3) to afford peptide 2 (23.5 mg, 67% yield). ¹H NMR (D₂O, 400 MHz), δ: 7.18-7.07 (m, 5H), 4.55 (dd, 1H), 4.45 (dd, 1H), 4.16 (dd, 1H), 4.07 (dd, 1H), 4.01 (d, 1H), 3.95 (s, 1H), 3.86 (s, 2H), 3.71 (dd, 1H), 3.56-3.52 (m, 2H), 3.39-3.26 (m, 4H), 3.04-2.99 (m, 4H), 2.90-2.70 (m, 3H), 2.53 (dd, 1H), 1.71-1.64 (m, 1H), 1.51-1.46 (m, 2H), 1.37-1.32 (m, 3H), 1.27-1.18 (m, 2H), 0.85-0.78 (m, 2H). ¹³C NMR (D₂O, 100 MHz), δ: 174.7, 174.5, 173.2, 172.9, 171.5, 171.4, 170.9, 170.7, 156.8, 136.1, 129.3, 128.9, 127.4, 78.1, 77.7, 73.5, 69.6, 68.0, 55.5, 55.1, 52.5, 52.1, 49.7, 43.6, 40.6, 40.4, 38.7, 37.1, 34.4, 30.0, 27.8, 27.4, 24.6, 22.6. ESI-MS: calculated for C₃₈H₅₄F₃N₁₃O₁₅: 989.38. found: 876.4 [M-CF₃COOH+H]⁺.

Synthesis of 3

To the solution of 1,8-diamino-3,6,10,13,16,19-hexaazabicyclo[6.6.6]-eicosine (1 mg, 2.47 μmol, 1 equiv) in 200 uL of sodium borate buffer (pH 8.5) was added S3 (2 mg, 2.88 μmol, 1.17 equiv) in 10 uL of DMSO. The mixture was incubated at room temperature for 2 h and purified by semi-preparative HPLC. The desired peak was collected and concentrated to afford 3 as a white powder (1.0 mg, 45%). ESI-MS: calculated for C₄₆H₇₂N₁₀O₈: 893.1.3. found: 894.0 [M+H]⁺.

Synthesis of 1

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To the solution of 3 (0.9 mg, 1.01 μmol, 1 equiv) in 30 uL of deionized water was added 2 (0.973 mg, 1.11 μmol, 1.1 equiv) in 20 uL of deionized water. The mixture was incubated at room temperature for 1 h and purified by semi-preparative HPLC. The desired peak was collected, concentrated, and lyophilized to afford 1 as a white powder (1.7 mg, 95%). ESI-MS: calculated for C₈₂H₁₂₅N₂₃O₂₁: 1769.01. found: 1770.21 [M+H]⁺, 885.43 [M+2H]²⁺, 590.90 [M+3H]³⁺.

Radiosynthesis of [⁶⁴Cu]3

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[⁶⁴Cu]Cu(OAc)₂ was prepared by adding 37-111 MBq of [⁶⁴Cu]CuCl₂ in 0.1 N HCl into 300 μL of 0.4 M ammonium acetate buffer (pH=5.5), followed by mixing and incubating for 15 min at room temperature. The [⁶⁴Cu]Cu(OAc)₂ solution (37-111 MBq) was then added into a solution of 3 (5 μg precursor per mCi ⁶⁴Cu) dissolved in 0.4 M NH₄OAc (pH=5.5) solution. The reaction mixture was incubated at 40° C. for 30 min. The labeled product was then purified by analytical HPLC. The radioactive peak containing desired product (Rt=17.67 min) was collected and concentrated by rotary evaporation to afford [⁶⁴Cu]3. The product was reconstituted in deionized water for use in following experiments. The specific activity of [⁶⁴Cu]3 was estimated to be 37 MBq nmol⁻¹.

Radiosynthesis of [⁶⁴Cu]1: “Click” Coupling of 2 and [⁶⁴Cu]3

To the solution of [⁶⁴Cu]3 (37 MBq) in 50 uL of deionized water was added 2 (1.14 equiv) in 50 uL of deionized water. The reaction mixture was incubated at 45° C. for 15 min to afford [⁶⁴Cu]1. The product was then reconstituted in 500-800 μL PBS, and passed through a 0.22-μm Millipore filter into a sterile dose vial for use. The specific activity of [⁶⁴Cu]1 was estimated to be 30 MBq

Experimental Methods

General Materials and Methods.

All chemicals and solvents were obtained from Aldrich (Milwaukee, Wis., USA) and used without further purification. 1,8-Diamino-3,6,10,13,16,19-hexaazabicyclo[6.6.6]-eicosine was purchased from Areva Med (Dallas, Tex.). ¹²⁵I-Echistatin was obtained from Perkin-Elmer (Waltham, Mass.). ¹H and ¹³C nuclear magnetic resonance (NMR) were recorded on a Varian 400 MHz spectrometer. Mass spectrum was obtained on a ThermoElectron Finnigan LTQ mass spectrometer equipped with an electrospray ionization (ESI) source (Thermo Scientific, USA). HPLC analyses were carried out on an analytical reversed-phase high performance liquid chromatography (HPLC) system equipped with a dual UV absorbance detector (Waters 2487) using a phenomenex C18 RP (250×4.6 mm 5 micron). The flow was 1 mL/min, with the mobile phase starting from 100% solvent A (0.1%© TFA in water), followed by a gradient mobile phase to 20% solvent A and 80%© solvent B (0.1% TFA in acetonitrile) at 30 min. The UV absorbance was monitored at 214 nm and the identification of the peptides was confirmed based on the UV spectrum using a PDA detector. The radioactivity was detected by a model of Ludlum 2200 single-channel radiation detector. The stability study was performed under the same HPLC condition. MicroPET scans were performed on a microPET R4 rodent model scanner (Siemens Medical Solutions USA, Inc., Knoxyille, Tenn.). The scanner has a computer-controlled bed and 10.8-cm transaxial and 8-cm axial fields of view (FOVs). It has no septa and operates exclusively in the 3-dimensional (3D) list mode. Animals were placed near the center of the FOV of the scanner.

Statistical Analysis.

Quantitative data were expressed as mean±SD. Means were compared using one-way ANOVA and student's t-test. P values<0.05 were considered statistically significant.

Partition Coefficient.

The partition coefficient value was expressed as log P. Log P of [⁶⁴Cu]1 was determined by measuring the distribution of radioactivity in 1-octanol and PBS. Approximately 111 kBq of [⁶⁴Cu]1 in 2 μL of PBS (pH=7.4) was added to a vial containing 0.5 mL 1-octanol and 0.5 mL of PBS (pH=7.4). After vigorously vortexing for 10 min, the vial was centrifuged at 12,500 rpm for 5 min to ensure the complete separation of layers. 200 μL of each layer was pipetted into test tubes, and radioactivity was measured using a gamma counter (Perkin-Elmer Packard Cobra). The mean value was calculated from triplicate experiments. The log P value of [⁶⁴Cu]1 was determined to be 4.94±0.10.

Probe Stability Determination. The stability of [⁶⁴Cu]1 was tested in PBS. In brief, 3.7 MBq of the [⁶⁴Cu]1 was pipetted into 0.5 mL of the PBS and incubated in PBS at room temperature. At various time points (1, 6, and 24 h), an aliquot of the solution was taken and the radiochemical purity was determined by reverse-phase HPLC under identical conditions. The parent [⁶⁴Cu]1 was determined to be >99%, 98%, and 96% at 1, 6, and 24 h, respectively.

Cell Culture.

U87MG human glioblastoma cell line was obtained from the American Type Culture Collection (ATCC, Manassas, Va.). U87MG glioma cells were grown in Dulbecco's modified medium (USC Cell Culture Core, Los Angeles, Calif.) supplemented with 10%© fetal bovine serum (FBS) at 37° C. in humidified atmosphere containing 5% CO₂.

Integrin α_(v)β₃ Receptor Binding Assay.

U87MG cells were suspended with binding buffer [25 mM 2-amino-2-(hydroxymethyl)-1,3-propanediol, hydrochloride (Tris-HCl), pH 7.4, 150 mM NaCl, 1 mM CaCl₂, 0.5 mM MgCl₂, 1 mM MnCl₂, and 0.1% bovine serum albumin (BSA)]. Incubation was conducted in a 96-well plate with a total volume of 200 μL in each well containing 2×10⁵ cells, 0.02 μCi (0.74 kBq) ¹²⁵I-echistatin (Perkin-Elmer), and 0-5000 nM of c-(RGDyK) or peptide 1 for 3 h on a shaker at room temperature. After incubation, cells were washed three times with cold phosphate buffered saline (PBS) with 0.1% BSA. Thereafter, the plate was heated to 40° C. and dried. The dried filter membranes were punched off from the wells and collected in polystyrene culture test tubes. Cell bound radioactivity was then measured using a gamma counter (Perkin-Elmer Packard Cobra). The IC₅₀ values were calculated by nonlinear regression analysis using the GraphPad Prism computer-fitting program (GraphPad Software, Inc., San Diego, Calif.). Each data point is a result of the average of duplicate wells.

Animal Model.

All animal studies were approved by the University of Southern California Institutional Animal Care and Use Committee. Female athymic nude mice (about 4-6 weeks old, with a body weight of 20-25 g) were obtained from Harlan Laboratories (Livermore, Calif.). The U87MG human glioma xenograft model was generated by subcutaneous injection of 5×10⁶ U87MG human glioma cells into the front flank of female athymic nude mice. The tumors were allowed to grow 3-5 weeks until 200-500 mm³ in volume. Tumor growth was followed by caliper measurements of the perpendicular dimensions.

MicroPET Imaging and Blocking Experiment.

MicroPET scans and imaging analysis were performed using a rodent scanner (microPET R4 scanner; Siemens Medical Solutions). About 7.4 MBq of [⁶⁴Cu]1 was intravenously injected into each mouse (n=3) under isoflurane anesthesia. Five-minute static scans were acquired at 2, 4, and 20 h pi. The images were reconstructed by a two-dimensional ordered-subsets expectation maximum (OSEM) algorithm. For each microPET scan, regions of interest were drawn over the tumor, normal tissue, and major organs on the decay-corrected whole-body coronal images. The radioactivity concentration (accumulation) within the tumor, muscle, liver, and kidneys were obtained from the mean value within the multiple regions of interest and then converted to % ID/g. For the blocking experiment, mice bearing U87MG tumors were scanned (5 min static) at 2, 4, and 20 h after the co-injection of 7.4 MBq of [⁶⁴Cu]1 with 10 mg/kg c(RGDyK) peptide per mouse.

Biodistribution Studies.

The U87MG tumor bearing mice (n=3) were injected with 7.4 MBq of [⁶⁴Cu]1. At 20 h after intravenous injection of [⁶⁴Cu]1, mice were sacrificed and dissected. Blood, U87MG tumor, major organs, and tissues were collected and weighed wet. The radioactivity in the tissues was measured using a gamma counter (Perkin-Elmer Packard Cobra). The results were presented as percentage injected dose per gram of tissue (% ID/g). For each mouse, the radioactivity of the tissue samples was calibrated with a known aliquot of the injected activity. Mean uptake (% ID/g) for a group of animals was calculated with standard deviations.

While various aspects and embodiments have been disclosed herein, other aspects and embodiments will be apparent to those skilled in the art. The various aspects and embodiments disclosed herein are for purposes of illustration and are not intended to be limiting, with the true scope and spirit being indicated by the following claims. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the method and compositions described herein. Such equivalents are intended to be encompassed by the following claims.

REFERENCES

The following references are incorporated herein in the entirety:

-   [1] Chen K and Chen X. Positron emission tomography imaging of     cancer biology: current status and future prospects. Semin Oncol     2011; 38:70-86. -   [2] Chen K and Conti P S. Target-specific delivery of peptide-based     probes for PET imaging. Adv Drug Deliv Rev 2010; 62:1005-22. -   [3] Zeglis B M and Lewis J S. A practical guide to the construction     of radiometallated bioconjugates for positron emission tomography.     Dalton Trans 2011; 40:6168-95. -   [4] Hao G, Singh A N, Oz O K, and Sun X. Recent advances in copper     radiopharmaceuticals. Curr Radiopharm 2011; 4:109-21. -   [5] Kolb H C and Sharpless K B. The growing impact of click     chemistry on drug discovery. Drug Discov Today 2003; 8:1128-37. -   [6] Nwe K and Brechbiel M W. Growing applications of “click     chemistry” for bioconjugation in contemporary biomedical research.     Cancer Biother Radiopharm 2009; 24:289-302. -   [7] Debets M F, van Berkel S S, Dommerholt J, Dirks A T, Rutjes F P,     and van Delft F L. Bioconjugation with strained alkenes and alkynes,     Ace Chem Res 2011; 44:805-15. -   [8] Haubner R, Beer A J, Wang H, and Chen X. Positron emission     tomography tracers for imaging angiogenesis. Eur J Nucl Med Mol     Imaging 2010; 37 Suppl 1:586-103. -   [9] Haubner R, Wester H J, Burkhart F, Senekowitsch-Schmidtke R,     Weber W, Goodman S L, et al. Glycosylated RGD-containing peptides:     tracer for tumor targeting and angiogenesis imaging with improved     biokinetics. J Nucl Med 2001; 42:326-36. -   [10] Liu W, Hao G, Long M A, Anthony T, Hsieh J T, and Sun X.     Imparting multivalency to a bifunctional chelator: a scaffold design     for targeted PET imaging probes. Angew Chem Int Ed Engl 2009;     48:7346-9. -   [11] Liu S, Li Z, Yap L P, Huang C W, Park R, and Conti P S.     Efficient preparation and biological evaluation of a novel     multivalency bifunctional chelator for 64Cu radiopharmaceuticals.     Chemistry 2011; 17:10222-5. -   [12] Debets M F, van Berkel S S, Schoffelen S, Rutjes F P, van Hest     J C, and van Delft F L. Aza-dibenzocyclooctynes for fast and     efficient enzyme PEGylation via copper-free (3+2) cycloaddition.     Chem Commun (Camb) 2010; 46:97-9. -   [13] Zhang X, Xiong Z, Wu Y, Cai W, Tseng J R, Gambhir S S, et al.     Quantitative PET imaging of tumor integrin alphavbeta3 expression     with 18F-FRGD2. J Nuel Med 2006; 47:113-21. 

What is claimed is:
 1. A compounds according to Formula III:

wherein L may be bound to any of the nitrogen atoms of the triazole moiety, wherein L includes at least one biological component, including without limitation a small molecule, peptide, proteins and/or nanoparticle, or alternatively, L is a chelator capable of coordinating a radiometal nuclide, the moiety M is optional, but when included may be one or more hydrophilic groups, including, for example, one or more polyethelene glycol units, one or more sugars, the moiety Q is a chelator capable of coordinating a radiometal nuclide, or alternatively, if L is a chelator, Q includes at least one biological component, including without limitation a small molecule, peptide, proteins and/or nanoparticle, and the benzene rings may be mono-, di-, tri-, or tetra- substituted with R₁ and R₂ groups selected from the group consisting of H, halide, hydroxy, alkyl, aryl, nitro or amine groups.
 2. A rapid, efficient, and catalyst-free click chemistry method for generating radiometal-labeled probes for imaging and therapy comprising: reacting an azido composition, N₃-L of Formula I with a strained alkyne of Formula III according to the following reaction:

wherein L may be bound to any of the nitrogen atoms of the triazole moiety, wherein L includes at least one biological component, including without limitation a small molecule, peptide, proteins and/or nanoparticle, or alternatively, L is a chelator capable of coordinating a radiometal nuclide, the moiety M is optional, but when included may be one or more hydrophilic groups, including, for example, one or more polyethelene glycol units, one or more sugars, the moiety Q is a chelator capable of coordinating a radiometal nuclide, or alternatively, if L is a chelator, Q includes at least one biological component, including without limitation a small molecule, peptide, proteins and/or nanoparticle, and the benzene rings may be mono-, di-, tri-, or tetra- substituted with R₁ and R₂ groups selected from the group consisting of H, halide, hydroxy, alkyl, aryl, nitro or amine groups.
 3. A cyclopeptide derivative of the following structure.

wherein at least on of moieties X, Y and Z includes a click chemistry linkage, such as a triazole moiety, wherein R is selected from the group consisting of a side chain of a natural amino acid and a side chain of an unnatural amino acid, wherein the natural amino acid and the unnatural amino acid is either in the D or L form; wherein X is a heterocycle or a linker comprising a hydrophilic moiety selected from the group consisting of hydroxyl, carbonyl, sulfonamide, sulfonate, phosphate, polar amino acid moiety, PEG moiety, sugar mimetic, and sugar moiety; wherein Y is a heterocycle, or a linker comprising a hydrophilic moiety selected from the group consisting of hydroxyl, carbonyl, sulfonamide, sulfonate, phosphate, polar amino acid moiety, PEG moiety, sugar mimetic, and sugar moiety; wherein Z is a heterocycle, or a linker comprising a hydrophilic moiety selected from the group consisting of hydroxyl, carbonyl, sulfonamide, sulfonate, phosphate, polar amino acid moiety, PEG moiety, sugar mimetic, and sugar moiety; wherein w is 1, 2, 3, 4, or 5; wherein any one of X, Y, and Z comprises a radiometal nuclide, including, but not limited to the following: ⁶¹Cu, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ⁶⁸Ga, ⁸⁶Y, and ⁸⁹Zr.
 4. The cyclopeptide of claim 3, wherein Y is a heterocycle; and X is a linker either comprising a sugar mimetic selected from the group consisting of a 4 to 6-membered carbocycle substituted with at least one hydroxyl group and a 5- to 6-membered heterocycle substituted with at least one hydroxyl group or comprising a sugar moiety selected from the group consisting of glucose and galactose.
 5. The cyclopeptide of claim 3, wherein: Y is a heterocycle; X is selected from the group consisting of:

Z is selected from the group consisting of:

wherein T is selected from the group consisting of:

wherein W is selected from the group consisting of:

wherein E is selected from the group consisting of:

each R is independently selected from the group consisting of a side chain of a natural amino acid and a side chain of an unnatural amino acid, wherein the natural amino acid and the unnatural amino acid is either in the D or L form; each R₃ is independently selected from the group consisting of —H, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ alkyloxy, aryl, aryl-(C₁-C₆ alkylene)-, 3- to 7-membered carbocycle, 3- to 7-membered heterocycle, hydroxy-C₁-C₆-alkyl, C₁-C₆-alkoxy-C₁-C₆-alkyl, and a PEG moiety, wherein the alkyl, alkenyl, alkynyl, alkoxy, aryl, carbocycle, and heterocycle groups are each optionally substituted; each R₄ is selected from the group consisting of —H, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ alkyloxy, aryl, aryl-(C₁-C₆ alkylene)-, 3- to 7-membered carbocycle, 3- to 7-membered heterocycle, hydroxy-C₁-C₆-alkyl, and C₁-C₆-alkoxy-C₁-C₆-alkyl, wherein the alkyl, alkenyl, alkynyl, alkyloxy, aryl, carbocycle, and heterocycle, groups are each optionally substituted; each R₅ is independently selected from the group consisting of —H, —OH, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ alkyloxy, aryl-(C₁-C₆ alkylene)-, hydroxy-C₁-C₆-alkyl, and C₁-C₆-alkoxy-C₁-C₆-alkyl, wherein the alkyl, alkenyl, alkynyl, alkyloxy, and aryl-alkylene groups are each optionally substituted; each v is 0, 1, 2, 3, or 4; m is 0, 1, 2, 3 or 4; p is an integer between 1 and 110; q is 1, 2, 3 or 4; r is 1, 2 or 3; r′ is 0 or 1; s is 1, 2, 3 or 4; and the radionuclide is selected from a radiometal nuclide, including, but not limited to the following: ⁶¹Cu, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ⁶⁸Ga, ⁸⁶Y, and ⁸⁹Zr. wherein the configuration of the chiral centers may be R or S or mixtures thereof.
 6. The cyclopeptide of claim 5, wherein: R is a side chain of a natural amino acid;

X is Y is 3a,8,9,13b-tetrahydro-3H-dibenzo[b,f][1,2,3]triazolo[4,5-d]azocine; and Z is selected from the group consisting of:

R₃ is selected from the group consisting of —H, C₁-C₆ alkyl, C₂-C₆ alkenyl, and C₂-C₆ alkynyl, wherein the alkyl, alkenyl, and alkynyl groups are each optionally substituted; and any one of X, Y, and Z comprises a radiometal nuclide including, but not limited to the following: ⁶¹Cu, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ⁶⁸Ga, ⁸⁶Y, and ⁸⁹Zr.
 7. The cyclopeptide of claim 3, wherein a portion of the peptide contains a primary amine group, such as a lysine residue, which is utilized for conjugation to moiety XYZ. The configuration of C-α of the amine-containing residue may be R or S.
 8. The cyclopeptide of claim 3, wherein the moiety XYZ contains zero, one, or more hydrophilic groups (such as hydroxyl, sulfonate, phosphate, etc.).
 9. The cyclopeptide of claim 3, wherein the nuclide label A is covalently conjugated to one or more of X, Y, and Z.
 10. The method according to claim 2, wherein the chelator is chelated to a radiometal nuclide which may be, but is not limited to the following: ⁶¹Cu, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ⁶⁸Ga, ⁸⁶Y, and ⁸⁹Zr.
 11. A method for preparing a radiopharmaceutical comprising: reacting an azido compound formula N₃-L with a strained alkyne to form of a 1,2,3-triazole under reaction conditions that do not include a catalyst.
 12. The method of claim 11, wherein L is cyclopeptide and the strained is capable of being coupled to a chelator.
 13. The method of claim 12, wherein the strained alkyne is coupled to a chelator prior to reaction with the azido compound to form an alkyne-containing chelator.
 14. The method of claim 13, wherein the alkyne-containing chelator is labeled by a radiometal prior to reaction with the azido compound.
 15. The method of claim 14, wherein the radiometal is selected from the group consisting of ⁶¹Cu, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ⁶⁸Ga, ⁸⁶Y, and ⁸⁹Zr.
 16. A method of modifying an RGD-sequence containing cyclic peptide with the moiety XYZ, which installs a nuclide label on the cyclic peptide, comprising: click chemistry. 